Neutropohil inhibitors

Abstract

Compositions enriched for Neutrophil Inhibitory Factor which inhibit neutrophil activity including adhesion to vascular endothelial cells are provided. Such compositions may comprise a glycoprotein isolated from nematodes. These compositions are useful in the therapy of conditions which involve abnormal or undesired inflammatory responses.

We claim:  
     
       1. An isolated DNA molecule which encodes a Neutrophil Inhibitory Factor comprising the amino acid sequence of FIG. 8 (SEQ. ID. NO. 33). 
     
     
       2. A method for producing a biologically active Neutrophil Inhibitory Factor which comprises the steps of culturing host cells having an expression vector which comprises a nucleic acid molecule which encodes a Neutrophil Inhibitory Factor wherein said Neutrophil Inhibitory Factor has neutrophil inhibitory activity and comprises an amino acid sequence of SEQ. ID. NO. 33 under conditions wherein said Neutrophil Inhibitory Factor is expressed, and isolating said Neutrophil Inhibitory Factor. 
     
     
       3. An isolated nucleic acid molecule which encodes a Neutrophil Inhibitory Factor (NIF) wherein said NIF comprises an amino acid sequence of SEQ. ID. NO. 33 and wherein said NIF has neutrophil inhibitory activity. 
     
     
       4. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a Neutrophil Inhibitory Factor which (i) occurs in or is isolated from an  Ancylostoma  species of parasitic worm or (ii) is obtainable from an Ancylostoma species of parasitic worm wherein said protein (a) has neutrophil inhibitory activity and (b) comprises the amino acid sequence of SEQ. ID. NO. 12. 
     
     
       5. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a Neutrophil Inhibitory Factor wherein said Neutrophil Inhibitory Factor (i) occurs in an Ancylostoma species of parasitic worm or is obtainable from an Ancylostoma species of parasitic worm (ii) has neutrophil inhibitory activity, and (iii) comprises an amino acid sequence which occurs in an Ancylostoma species of parasitic worm selected from the group consisting of: 
       (1) His-Asn-Gly-Tyr-Arg-Ser-X 1 -Leu-Ala-Leu-Gly-His-X 2 -X 3 -Ile-X 4  (SEQ. ID. NO. 1) , wherein X 1  is Arg, Lys, or Asn; X 2  is Ile or Val; X 3  is Ser or Gly; and X 4  is Thr or Ser;  
       (2) X 5 -Ala-Pro-X 6 -Ala-Ser-Lys-Mat-Arg-Tyr-X 7 -X 8 -Tyr-Asp-Cys-X 9 -X 10 -Glu-X 11 -Ser-Ala-Tyr (SEQ. ID. NO. 2), wherein X 5  is Phe or Tyr; X 6  is Arg, Ser, or Thr; X 7  is Leu or Met; X 8  is Glu or Lys; X 9  is Glu or Asp; X 10  is Ala or Ser; and X 12  is Lys or Arg;  
       (3) Gly-Glu-Gly-Val-Leu-Tyr-Arg-Ser (SEQ. ID. NO. 3);  
       (4) Ile-Ser-Asn-Phe-Ala-Asn-Leu-Ala-Trp-Asp-X 12 -Arg-Glu-Lys-X 13 -Gly-Cys-Ala-Val-X 14  (SEQ. ID. NO. 4), wherein X 12  is Thr or Ala; X 13  is Phe or Val; and X 14  is Val or Ala; and  
       (5) His-Val-Val-Cys-His-Tyr-Pro-Lys (SEQ. ID. No. 5).  
     
     
       6. The nucleic acid molecule of any of  claim 3 ,  4  or  5  further comprising a promoter operably linked to the nucleic acid molecule. 
     
     
       7. An expression vector comprising the nucleic acid molecule of any of  claim 3 ,  4  or  5  operably linked to control sequences recognized by a host cell transformed with the vector. 
     
     
       8. A host cell transformed with the vector of  claim 7 . 
     
     
       9. An isolated nucleic acid molecule comprising the nucleotide sequence of SEQ. ID. NO. 32. 
     
     
       10. An isolated nucleic acid molecule comprising the nucleotide sequence of SEQ. ID. NO. 40. 
     
     
       11. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the amino acid sequence of SEQ. ID. NO. 33. 
     
     
       12. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the amino acid sequence of SEQ. ID. NO. 41. 
     
     
       13. An expression vector comprising an isolated nucleic acid molecule having a nucleic acid sequence consisting essentially of the nucleic acid sequence of FIG. 8 (SEQ. ID. NO. 32). 
     
     
       14. An expression vector comprising an isolated nucleic acid molecule having a nucleic acid sequence consisting essentially of the nucleic acid sequence of SEQ. ID. NO. 40. 
     
     
       15. An expression vector comprising an isolated nucleic acid molecule having a nucleic acid sequence which encodes an amino acid sequence consisting essentially of the amino acid sequence of FIG. 8 (SEQ. ID. NO. 33). 
     
     
       16. An expression vector comprising an isolated nucleic acid molecule having a nucleic acid sequence which encodes an amino acid sequence consisting essentially of the amino acid sequence of SEQ. ID. NO. 41. 
     
     
       17. An expression vector comprising an isolated nucleic acid molecule having the nucleotide sequence of SEQ. ID. NO. 32. 
     
     
       18. An expression vector comprising an isolated nucleic acid molecule having the nucleotide sequence of SEQ. ID. NO. 40. 
     
     
       19. An expression vector comprising an isolated nucleic acid molecule having a nucleotide sequence which encodes the amino acid sequence of SEQ. ID. NO. 33. 
     
     
       20. An expression vector comprising an isolated nucleic acid molecule having a nucleotide sequence which encodes the amino acid sequence of SEQ. ID. NO. 41. 
     
     
       21. A host cell which contains the expression vector or any of  claims 13  to  20 . 
     
     
       22. The host cell of  claim 21  which is a CHO cell. 
     
     
       23. An isolated nucleic acid molecule encoding an amino acid sequence for a Neutrophil Inhibitory Factor (NIF) wherein said NIF: 
       (a) occurs in an Ancylostoma species or is obtainable from a NIF which occurs in an Ancylostoma species;  
       (b) has neutrophil inhibitory activity; and  
       (c) comprises an amino acid sequence selected from the group consisting of SEQ. ID. NOs. 1, 2, 3, 4, and 5.  
     
     
       24. An isolated nucleic acid molecule which encodes an amino acid sequence for a Neutrophil Inhibitory Factor (NIP) where said NIF: 
       (a) is obtainable from an Ancylostoma species;  
       (b) has neutrophil inhibitory activity; and  
       (c) comprises an amino acid sequence selected from the group consisting of SEQ. ID. NOs. 1, 2, 3, 4, and 5 which occurs in an Ancylostoma species.  
     
     
       25. An isolated nucleic acid molecule which encodes an amino acid sequence for a Neutrophil Inhibitory Factor (NIF) where said NIF: 
       (a) is obtainable from an Ancylostoma species;  
       (b) has neutrophil inhibitory activity; and  
       (c) comprises SEQ. ID. NOs. 1 to 5.  
     
     
       26. An isolated nucleic acid molecule which encodes an amino acid sequence for Neutrophil Inhibitory Factor (NIF) wherein said NIF: 
       (a) is isolated from an Ancylostoma species;  
       (b) bas neutrophil inhibitory activity; and  
       (c) comprises an amino acid sequence selected from the group consisting of  
       (i) His-Asn-Gly-Tyr-Arg-Ser-X 1 -Leu-Ala-Leu-Gly-His-X 2 -X 3 -Ile-X 4  (SEQ. ID. NO. 1), wherein X 1  is Arg, Lys, or Asn; X 2  is Ile or Val; X 3  is Ser or Gly; and X 4  is Thr or Ser;  
       (ii) X 5 -Ala-Pro-X 6 -Ala-Ser-Lya-Mat-Arg-Tyr-X 7 -X 8 -Tyr-Asp-Cys-X 9 -X 10 -Glu-X 11 -Ser-Ala-Tyr (SEQ. ID. NO. 2), wherein X 5  is Phe or Tyr; X 6  is Arg, Ser, or Thr; X 7  is Leu or Met; X 8  is Glu or Lys; X 9  is Glu or Asp; X 10  is Ala or Ser; and X 11  is Lye or Arg;  
       (iii) Gly-Glu-Gly-Val-Leu-Tyr-Arg-Ser (SEQ. ID. NO. 3);  
       (iv) Ile-Ser-Asn-Phe-Ala-Asn-Leu-Ala-Trp-Asp-X 12 -Arg-Glu-Lye-X 13 -Gly-Cys-Ala-Val-X 14  (SEQ. ID. NO. 4), wherein X 12  is Thr or Ala; X 13  is Phe or Val; and X 14  is Val or Ala; and  
       (v) His-Val-Val-Cys-His-Tyr-Pro-Lys (SEQ. ID. NO. 5).  
     
     
       27. An isolated nucleic acid molecule which encodes a Neutrophil Inhibitory Factor (NIF) wherein said NIF: 
       (a) occurs in or is obtainable from an Ancylostoma species;  
       (b) has neutrophil inhibitory activity;  
       (c) includes an amino acid sequence selected from the group consisting of  
       (i) His-Asn-Gly-Tyr-Arg-Ser-X 1 -Leu-Ala-Lau-Gly-His-X 2 -X 3 -Ile-X 4  (SEQ. ID. NO. 1), wherein X 1  is Arg, Lys, or Asn; X 2  is Ile or Val; X 3  is Ser or Gly; and X 4  is Thr or Ser;  
       (ii) X 5 -Ala-Pro-X 6 -Ala-Ser-Lys-Met-Arg-Tyr-X 7 -X 8 -Tyr-Asp-Cys-X 9 -X 10 -Glu-X 11 -Ser-Ala-Tyr (SEQ. ID. NO. 2), wherein X 5  is Phe or Tyr; X 6  is Arg, Ser, or Thr; X 7  is Leu or Met; X 8  is Glu or Lys; X 9  is Glu or Asp; X 10  is Ala or Ser; and X 11  is Lys or Arg;  
       (iii) Gly-Glu-Gly-Val-Leu-Tyr-Arg-Ser (SEQ. ID. NO. 3);  
       (iv) Ile-Ser-Asn-Phe-Ala-Asn-Leu-Ala-Trp-Asp-X 12 -Arg-Glu-Lys-X 13 -Gly-Cys-Ala-val-X 14  (SEQ. ID. NO. 4), wherein X 12  is Thr or Ala; X 13  is Phe or Val; and X 14  is Val or Ala; and  
       (v) His-Val-Val-Cya-His-Tyr-Pro-Lys (SEQ. ID. NO. 5;  
       which sequence is obtainable from an Ancylostoma species. 
     
     
       28. An isolated nucleic acid molecule which encodes an amino acid sequence for Neutrophil Inhibitory Factor (NIF) wherein said NIF: 
       (a) has neutrophil inhibitory activity; and  
       (b) comprises the following amino acid sequences  
       (i) His-Asn-Gly-Ty-Arg-Ser-X 1 -Leu-Ala-Leu-Cly-gis-X 2 -X 3 -Ile-X 4  (SEQ. ID. NO. 1), wherein X 1  is Arg, Lys, or Asn; X 2  is Ile or Val; X 3  is Ser or Gly; and X 4  is Thr or Ser;  
       (ii) X 5 -Ala-Pro-X 6 -Ala-Ser-Lys-Met-Arg-Tyr-X 7 -X 8 -Tyr-Asp-Cys-X 9 -X 10 -Glu-X 11 -Ser-Ala-Tyr (SEQ. ID. NO. 2), wherein X 5  is Phe or Tyr; X 6  is Arg, Ser, or Thr; X 7  is Leu or Met; X 8  is Glu or Lys; X 9  is Glu or Asp; X 10  is Ala or Ser; and X 11  is Lys or Arg;  
       (iii) Gly-Glu-Gly-Val-Leu-Tyr-Arg-Ser (SEQ. ID. NO. 3);  
       (iv) Ile-Ser-Asn-Phe-Ala-Asn-Leu-Ala-Trp-Asp-X 12 -Arg-Glu-Lys-X 13 -Gly-Cys-Ala-Val-X 14 , (SEQ ID. NO. 4), wherein X 12  is Thr or Ala; X 13  is Phe or Val; and X 14  is Val or Ala; and  
       (v) His-Val-Val-Cys-His-Tyr-Pro-Lys (SEQ. ID. NO. 5);  
       which sequences are obtainable from an Ancylostoma species. 
     
     
       29. An isolated nucleic acid molecule which encodes an amino acid sequence for Neutrophil inhibitory Factor (NIF) wherein said NIF has neutrophil inhibitory activity; and wherein said nucleic acid molecule 
       (a) is obtainable from an Ancylostoma species; and  
       (b) encodes an amino acid sequence selected from the group consisting of  
       (i) His-Asn-Gly-Tyr-Arg-Ser-X 1 -Leu-Ala-Leu-Gly-His-X 2 -X 3 -Ile-X 4  (SEQ. ID. NO. 1), wherein X 1  is Arg, Lys, or Asn; X 2  is Ile or Val; X 3  is Ser or Gly, and X 4  is Thr or Ser;  
       (ii) X 5 -Ala-Pro-X 6 -Ala-Ser-Lys-Met-Arg-Tyr-X 7 -X 8 -Tyr-Asp-Cys-X 9 -X 10 -Glu-X 11 -Ser-Ala-Tyr (SEQ. ID. NO. 2), wherein X 5  is Phe or Tyr; X 6  is Arg, Ser, or Thr; X 7  is Leu or Met; X 8  is Glu or Lys; X 9  is Glu or Asp; X 10  is Ala or Ser; and X 11  is Lys or or Arg;  
       (iii) Gly-Glu-Gly-val-Leu-Tyr-Arg-Ser (SEQ. ID. NO. 3);  
       (iv) Ile-Ser-Asn-Phe-Ala-Asn-Leu-Ala-Trp-Asp-X 12 -Arg-Glu-Lys-X 13 -Gly-Cys-Ala-Val-X 14  (SEQ. ID. NO. 4), wherein X 12  is Thr or Ala; X 13  is Phe or Val; and X 14  is Val or Ala; and  
       (v) His-Val-Val-Cys-His-Tyr-Pro-Lys (SEQ. ID. NO. 5).  
     
     
       30. An isolated nucleic acid molecule which encodes an amino acid sequence for Neutrophil Inhibitory Factor (NIF) wherein said NIF has neutrophil inhibitory activity; and wherein said isolated nucleic acid 
       (a) is isolated from an Ancylostema species; and  
       (b) encodes an amino acid sequence selected from the group consisting of  
       (i) His-Asn-Gly-Tyr-Arg-Ser-X 1 -Leu-Ala-Leu-Gly-His-X 2 -X 3 -Ile-X 4  (SEQ. ID. NO. 1), wherein X 1  is Arg, Lys, or Asn; X 2  is Ile or Val; X 3  is Ser or Gly; and X 4  is Thr or Ser;  
       (ii) X 5 -Ala-Pro-X 6 -Ala-Ser-Lys-Met-Arg-Tyr-X 7 -X 8 -Tyr-Asp-Cys-X 9 -X 10 -Glu-X 11 -Ser-Ala-Tyr (SEQ. ID. NO. 2), wherein X 5  is Phe or Tyr; X 6  is Arg, Ser, or Thr; X 7  is Leu or Met; X 8  is Glu or Lys; X 9  is Glu or Asp; X 10  is Ala or Ser; and X 11  is Lys or Arg;  
       (iii) Gly-Glu-Gly-Val-Leu-Tyr-Arg-Ser (SEQ. ID. No. 3);  
       (iv) Ile-Ser-Asn-Phe-Ala-Asn-Leu-Ala-Trp-Asp-X 2 -Arg-Glu-Lye-X 13 -Gly-Cys-Ala-Val-X 14  (SEQ. ID. NO. 4), wherein X 12  is Thr or Ala, X 13  is Phe or Val; and X 14  is Val or Ala; and  
       (v) His-Val-Val-Cys-His-Tyr-Pro-Lys (SEQ. ID. NO. 5).  
     
     
       31. An expression vector comprising an isolated nucleic acid molecule of any of  claims 23  to  30  operably linked to control sequences recognized by a host cell transformed with the vector. 
     
     
       32. A host cell transformed with an expression vector of  claim 31 . 
     
     
       33. A method of making Neutrophil Inhibitory Factor comprising the steps of culturing host cells having an expression vector which comprises an isolated nucleic acid molecule which encodes an amino acid sequence for a Neutrophil Inhibitory Factor inserted in said cells under cell culture conditions whereby said Neutrophil Inhibitory Factor is expressed and isolating Neutrophil Inhibitory Factor from the host cells, wherein said isolated nucleic acid molecule is selected from the isolated nucleic acid molecules of  claims 23  to  30 . 
     
     
       34. A method of making Neutrophil Inhibitory Factor comprising the steps of culturing host cells having an expression vector which comprises an isolated nucleic acid molecule which encodes an amino acid sequence for a Neutrophil Inhibitory Factor inserted in said cells under cell culture conditions whereby the Neutrophil Inhibitory Pactor is expressed and said Neutrophil Inhibitory Factor is secreted and isolating the Neutrophil Inhibitory Factor secreted by the host cells, wherein said isolated nucleic acid molecule is selected from the isolated nucleic acid molecules of  claims 23  to  30 .